This invention relates generally to testing of biological fluids such as blood, urine, and the like, more particularly to immunoassays which employ antibodies to detect antigens.
Immunochromatographic strip formats have become increasingly popular for qualitative and semi-quantitative assays, particularly those which use visual detection schemes. This type of immunoassay involves the application of a liquid test sample suspected of containing an analyte to an application zone of an immuno-chromatographic strip. The presence of the analyte is typically indicated by a color which develops at a particular region of the strip. For example, pregnancy tests in which the analyte is human chronic gonadotropin (hCG).
Generally, a sample is brought into contact with an antibody to the analyte (i e, an anti-analyte antibody) which binds to the analyte, the antibody being labeled with a detectable moiety which, after separating the bound and free portions of the antibody, is used to indicate the amount of the analyte present. In order to collect the analyte bound to the antibody, a second anti-analyte antibody binding to a second epitope of the analyte may be immobilized on a test strip to capture the analyte bound to the first antibody. If the second anti-analyte antibody is not immobilized on a test strip, a pair of binding partners, typically a substance and its antibody, may be used to collect the second anti-analyte antibody. The first part of the binding pair is attached to the second anti-analyte antibody and the second part of the binding pair is immobilized at a specific location in a test strip to bind to the first part of the pair, thus immobilizing the analyte bound to the labeled first anti-analyte antibody so that it can be measured.
Usually, a test strip is comprised of a matrix material through which the test fluid, which may have the analyte suspended or dissolved therein, can flow by capillarity from the application zone to a detection zone where a detectable signal, or absence thereof, reveals the presence of the analyte. Typically, the strip will include means for immunospecifically binding the analyte to be detected with its specific binding partner which bears a detectable label. In one such scheme, as disclosed in U.S. Pat. No. 4,446,232, the strip contains an enzyme labeled, mobile binding partner for the analyte which is in a zone downstream from the sample application zone. If analyte is present in the test sample, it will combine with its labeled binding partner to form a complex which can flow along the strip to a detection zone which contains a substrate for the enzyme label capable of providing a colored response in the presence of the enzyme label. The strip also contains a zone in which analyte has been immobilized, so that the labeled binding partner which does not combine with the analyte, due to absence of analyte in the sample, will be captured and thereby prevented from reaching the detection zone. There have been published various modifications of this technique, all of which involve some specific binding system in which the presence or absence of analyte in the test sample is determined by the detection or lack thereof of labeled binding partner in the detection zone. In U.S. Pat. No. 4,868,108 there is disclosed a similar scheme with the addition of an immobilized capture reagent for the enzyme labeled binding partner in the detection zone to concentrate the enzyme label and enhance its ability to react with the enzyme substrate to thereby render the assay more sensitive.
Not all of the schemes for immunochromatography rely on an enzyme labeled binding partner/enzyme substrate to provide the signal for detection of the analyte. In U.S. Pat. No. 4,806,311 there is disclosed a multi zone test device for the specific binding assay determination of an analyte and an immobilized binding partner therefore together with a detection zone for receiving labeled reagent which migrates thereto from the reagent zone. The detection zone contains an immobilized form of a binding substance for the labeled reagent. The labeled reagent bears a chemical group having a detectable physical property, so that it does not require a chemical reaction with another substance. Exemplary of such groups are colored species, fluorescers, phosphorescent molecules, radioisotopes and electroactive moieties U.S. Pat. No. 4,313,734 describes the use of gold sols as labels for antibodies which are detectable without a chemical change.
The present invention relates to a method of overcoming interference with the results of immunoassays by non-specific binding of heterophilic antibodies in the sample to the labeled antibody. It was found that false positive results were occurring because an unbound labeled antibody was being retained in the detection region where it was expected that only those labeled antibodies which had bound to the target antigen (i e, the analyte) would be found. That is, the measurement of the amount of the bound antibody by using the label indicated the presence of the antigen, when none were present. Displacing the non-specific binding of the heterophilic anti-bodies was possible, but this created another difficulty, which was overcome by the present inventors, as will be seen in the description below.